The broad objective of the proposed research is to obtain a molecular-level understanding of the interactions between transcriptional activators and their target proteins within the transcriptional machinery using a suite of chemical approaches. Misregulation of transcription is either a cause or an effect of human diseases such as cancer, metabolic disorders, and infectious diseases. Thus, molecules that inhibit or promote the protein-protein interactions that regulate transcription are highly desirable both as mechanistic probes and, in the long term, as therapeutic agents that correct transcriptional errors. Only a handful of such molecules have been reported, however, due in part to the still-limited understanding of the mechanism of transcriptional regulation. In the case of transcriptional activators, proteins that activate the transcription of particular genes in a signal- responsive fashion, little conclusive is known about either the interaction partners within the transcriptional machinery or the functionally relevant thermodynamic parameters. As part of Specific Aim 1, we will kinetically characterize the interactions between several transcriptional activators and their target proteins in order to parse the connection between levels of gene up-regulation and the rates of association and dissociation of activator-transcriptional machinery protein complexes. In Specific Aims 2 and 3 in vivo (S. cerevisiae) crosslinking experiments along with FT-ICR mass spectrometric analysis of crosslinking products will be employed to identify the physiologically relevant targets of transcriptional activators, thus addressing a long-standing and fundamental mechanistic question in eukaryotic transcription. Taken together, the data generated through these experiments will provide a high resolution map of what and where transcriptional activators bind in the transcriptional machinery as they initiate gene up-regulation. This information will be enormously enabling for the discovery of transcription-targeting small molecules and, longer term, for the development of small molecules that target individual transcriptional activator-transcriptional machinery protein interactions implicated in human disease. PUBLIC HEALTH RELEVANCE: The experiments outlined in this proposal will provide a detailed picture of the binding profile of transcriptional activators and the contribution that the individual binding events make to transcriptional regulation. These data will make it possible to design more effective screens of small molecules that function as activators or inhibitors of transcription and, further, will provide a strategy by which specificity can be quickly evaluated. Ultimately, this will facilitate the discovery and development of transcription-based therapeutics.